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Spectrochimica Acta Part A 78 (2011) 1295–1301

Contents lists available at ScienceDirect

Spectrochimica Acta Part A: Molecular andBiomolecular Spectroscopy

journa l homepage: www.e lsev ier .com/ locate /saa

tudies on the interaction of salvianolic acid B with human hemoglobin byulti-spectroscopic techniques

ingting Chena, Shajun Zhub, Hui Caoa, Yanfang Shanga, Miao Wanga,uoqing Jianga, Yujun Shia,∗, Tianhong Luc

School of Chemistry and Chemical Engineering, Nantong University, Nantong 226019, 9 Seyuan Road, PR ChinaDepartment of General Surgery, Affiliated Hospital of Nantong University, Nantong 226001, PR ChinaCollege of Chemistry and Material Science, Nanjing Normal University, Nanjing 210097, PR China

r t i c l e i n f o

rticle history:eceived 8 December 2010eceived in revised form7 December 2010

a b s t r a c t

The interaction between salvianolic acid B (Sal B) and human hemoglobin (HHb) under physiologicalconditions was investigated by UV–vis absorption, fluorescence, synchronous fluorescence and circulardichroism spectroscopic techniques. The experimental results indicate that the quenching mechanismof fluorescence of HHb by Sal B is a static quenching procedure, the binding reaction is spontaneous, and

ccepted 28 December 2010

eywords:alvianolic acid Buman hemoglobin

nteraction

the hydrophobic interactions play a major role in binding of Sal B to HHb. Based on Förster’s theory ofnon-radiative energy transfer, the binding distance between Sal B and the inner tryptophan residues ofHHb was determined to be 2.64 nm. The synchronous fluorescence experiment revealed that Sal B cannot lead to the microenvironmental changes around the Tyr and Trp residues of HHb, and the bindingsite of Sal B on HHb is located at �1�2 interface of HHb. Furthermore, the CD spectroscopy indicated the

b is

luorescenceircular dichroism

secondary structure of HH

. Introduction

Salvia miltiorrhiza (Danshen) is a well-known Chinese tradi-ional herb medicine, used clinically for treatment of coronary heartisease [1], cerebrovascular disease [2], hepatitis [3], and hepato-irrhosis [4]. Salvianolic acid B (Sal B, Fig. 1) is one of the majorater-soluble components extracted from S. miltiorrhiza, and is

lso the most abundant and bioactive ingredient of the salviano-ic acids in danshen [5]. It was reported that Sal B could reducehe atherosclerosis [6], improve the regional cerebral blood flow7], prohibit the platelet aggregation [8], ameliorate the hemorhe-logy parameters [9] and protect against ischemia–reperfusionnjuries in heart and brain by reducing lipid peroxides, scav-nging free radicals and improving the energy metabolism10].

Hemoglobin (Hb) is the iron-containing oxygen-transport met-lloprotein in the red blood cells. As a carrier of oxygen, hemoglobinransports oxygen from the lungs or gills to the rest of the body

here it releases the oxygen for cell use. It also aids, both directly

nd indirectly, the transport of carbon dioxide and regulates theH of blood [11]. It removes hydrogen ions in the capillaries andarries them to the lungs. In addition, it is involved in many

∗ Corresponding author. Tel.: +86 513 85012851; fax: +86 513 85012851.E-mail address: yjshi2001@yahoo.com.cn (Y. Shi).

386-1425/$ – see front matter © 2011 Elsevier B.V. All rights reserved.oi:10.1016/j.saa.2010.12.081

not changed in the presence of Sal B.© 2011 Elsevier B.V. All rights reserved.

clinical diseases such as leukemia, anemia, heart disease, and exces-sive loss of blood [12]. Hemoglobin exists as a tetramer of globinchains that is composed of two � and two � subunits. The �subunit contains 141 amino acid residues whereas � subunit con-tains 146 amino acid residues. There are two types of subunitcontacts in a Hb molecular, i.e. �1�1 (or its �2�2 symmetry equiv-alent) and �1�2 (or its �2�1 symmetry equivalent) [13]. Eachsubunit has one redox iron heme as its prosthetics group, andthe heme is located in the crevices at the exterior of the subunit[14].

Transportation, distribution, metabolism and bioavailability ofmany drugs in vivo are closely related to their binding withproteins, hence investigating the mechanism of drug–proteininteraction is of crucial importance for us to understand the phar-macodynamics and pharmacokinetics behavior of the drug. HHbis the major hemoprotein of red blood cell, which can reversiblybind with many kinds of endogenous and exogenous agents suchas many drugs [15–17]. Although the study of Sal B’s various phar-macological effects has been described well, the interaction of SalB with HHb has not been investigated. The binding study of SalB and HHb is of toxicological and medical importance, and our

work should be valuable in chemistry, life sciences and clinicalmedicine.

In the present work, we studied in vitro interaction of SalB with HHb under the simulative physiological conditions usingUV–visible absorption spectroscopy, fluorescence spectroscopy,

1 a Acta Part A 78 (2011) 1295–1301

sstsSm

2

2

wL(T(0iHopbrood

2

(stwiflsSmwor

2

recm

2

rtq5sts

OH

HO

O

O

O

O

OH

OH

COOH

O COOH

OH

OH

that the microenvironment around the aromatic acid residues wasnot exposed to any change upon Sal B–HHb complexation. Fur-thermore, there is no obviously spectral change of Soret band inthe presence of Sal B, indicating that all four heme groups boundto hemoglobin are not directly attacked and degraded by Sal B,

500450400350300250200

0.0

0.5

1.0

1.5

2.0

2.5

3.0

Ab

so

rban

ce

a: [HHb]

b: [Sal B]

c: [HHb+Sal B]-[Sal B]

a

b

c

296 T. Chen et al. / Spectrochimic

ynchronous fluorescence spectroscopy and circular dichroismpectroscopy. The binding mechanism of Sal B with HHb (associa-ion constants, thermodynamic parameters, the number of bindingites, the binding forces, and the energy transfer distance betweenal B and HHb) was estimated. The effect of Sal B on HHb confor-ation was also discussed.

. Materials and methods

.1. Materials

Human hemoglobin (HHb, catalogue number H7379)as purchased from Sigma–Aldrich Chemical Company (St.

ouis, MO, USA) and used without further purification. Sal B≥98.0%) was obtained from Zelang Co. Ltd. (Nanjing, China).ris(hydromethyl)aminomethane (Tris) was purchased from AcrosGeel, Belgium). The Tris–HCl buffer (0.05 M, pH 7.40) containing.10 M NaCl was selected to keep the pH value and maintain the

onic strength of the solution. The 1.25 × 10−5 M stock solution ofb was prepared in pH 7.40 Tris–HCl buffer. The stock solutionf Sal B (1.25 × 10−4 M) was prepared by dissolving Sal B withH 7.40 Tris–HCl buffer. For CD spectra, the sodium phosphateuffer solution (20 mM, pH 7.40) was selected according to theequirement of the apparatus. All other reagents and solvents weref analytical grade and used without further purification unlesstherwise noted. All aqueous solutions were prepared using newlyouble-distilled water.

.2. Fluorescence spectra

Fluorescence spectra were recorded on a RF-5301PC fluorometerShimadzu, Japan) equipped with 1.0 cm quartz cell. The emissionpectra were recorded between 290 and 550 nm with an exci-ation wavelength of 280 nm. Excitation and emission slit widthere 5 nm and 3 nm, respectively. A series of assay samples were

ncubated for 1 h at 291 and 300 K, respectively, and then theuorescence intensities were recorded. Synchronous fluorescencepectra of HHb in the absence and presence of different amounts ofal B were also recorded by scanning the excitation and emissiononochromator simultaneously. The wavelength interval (��)as fixed individually at 15 and 60 nm, at which the spectrum

nly showed the spectroscopic behavior of tyrosine and tryptophanesidues of HHb, respectively.

.3. UV absorption spectra

The ultraviolet–visible (UV–vis) absorption spectra wereecorded on a UV-2450 spectrophotometer (Shimadzu, Japan)quipped with 0.5 cm quartz cells. The pH measurements werearried out on a PHS-3C Exact Digital pH meter (Cole-Parmer Instru-ent Co., IL, USA).

.4. CD spectra

CD spectra of the samples in the range of 190–250 nm wereecorded on a JASCO J-810 automatic recording spectropolarime-er (Tokyo, Japan) controlled by the Jasco software with a 0.1 cm

uartz cell at room temperature, and the speed of scanning was0 nm/min. The concentration of HHb was 5 �M, and the bufferolution was selected as the blank and was automatically sub-racted from each spectrum during scanning. Each sample wascanned for three times to average for a CD spectrum.

HO

Fig. 1. Molecular structure of salvianolic acid B.

3. Results and discussion

3.1. UV–vis absorption spectra studies

UV–vis absorption measurement is a simple but efficaciousmethod to explore the structural changes of protein and to inves-tigate protein–ligand complex formation. In this study, we usedthe difference absorption spectroscopy to obtain spectra. UV–visabsorption spectra of HHb in the absence and presence of Sal Bwere obtained by subtracting corresponding the spectrum of Sal Bin the buffer from that of Sal B–HHb complex. The UV–vis absorp-tion spectra of HHb without and with Sal B are shown in Fig. 2.It can be seen that HHb has three absorption peaks. The strongabsorption peak at 210 nm reflects the amide bonds of the protein.The weak absorption peak at 278 nm appears due to the aromaticamino acids (Trp, Tyr and Phe). The peak at 405 nm correspondsto the porphyrin-Soret band of HHb [18]. With addition of Sal Bto HHb solution, the intensity of the peak at 210 nm decreaseswith a slightly red shift, indicating disturbances to the microen-vironment around the amide bonds in the protein. The resultsindicate that there exists interaction between Sal B and HHb andground state complex is formed. The position and absorbance ofthe peak at 278 nm does not change significantly, which shows

Wavelength (nm)

Fig. 2. UV–visible spectra of HHb in the presence of Sal B (T = 291 K, pH 7.4):(a) absorption spectrum of HHb only; (b) absorption spectrum of Sal B only;(c) difference absorption spectrum between Sal B–HHb and Sal B. CHHb = 5.0 �M,CSal B = 15.0 �M.

T. Chen et al. / Spectrochimica Acta

550500450400350300

0

100

200

300

400

500F

luo

rescen

ce In

ten

sit

y

Wavelength (nm)

a

k

a

k

FH2

ag

3

itmttiattscaovatisOtetcueipBb4i[e

3

ic

ig. 3. Effect of Sal B on fluorescence spectra of HHb (T = 291 K, pH 7.4): (a) 5.0 �MHb; (b–k) 5.0 �M HHb in the presence of 2.5, 5, 7.5, 10, 12.5, 15, 17.5, 20.0, 22.5,5 �M Sal B.

nd the interaction does not affect the structure of the hemeroup.

.2. Fluorescence quenching of HHb by Sal B

For macromolecules, the fluorescence measurements can givenformation of the binding of small molecule substances to pro-ein at the molecular level, such as the binding mechanism, binding

ode, binding constants, intermolecular distances, etc. HHb con-ains three Trp residues in each �� dimer, for a total of six in theetramer: two �-14 Trp, two �-15 Trp, and two �-37 Trp [19]. Thentrinsic fluorescence of HHb primarily originates from �-37 Trpt the �1�2 interface, though it may contain some contribution byhe surface Trp residues, �-14 and �-15 Trp [20]. Like most pro-eins, the characteristic of the intrinsic fluorescence of HHb is veryensitive to its microenvironment. Some factors such as proteinonformational transitions, subunit association, substrate bindingnd denaturation, can result in the intrinsic fluorescence changesf protein [21]. Thus, the intrinsic fluorescence of proteins can pro-ide considerable information about their structure and dynamics,nd is often used to study the protein folding and association reac-ions. The effect of Sal B on HHb fluorescence intensity is shownn Fig. 3. As seen in Fig. 3, HHb had a strong fluorescence emis-ion band at 332 nm by fixing the excitation wavelength at 280 nm.bviously, the addition of Sal B to HHb caused a gradual decrease in

he fluorescence intensity of HHb without changing the maximummission wavelength and shape of the peaks, which is interpretedhat Sal B could interact with HHb and quench its intrinsic fluores-ence. No spectral shifting was observed for the emission spectrapon Sal B–HHb complexation, indicating that Trp residues are notxposed to any change in polarity [22]. The fluorescence quench-ng result suggests that the binding site of Sal B on HHb was closeroximity to �-37 Trp residue of HHb. Subsequent addition of Salprovoked an increase in the emission at ∼486 nm due to Sal B

ound to the HHb. Moreover, the occurrence of isoactinic point at36 nm might also indicate the existence of bound and free Sal B

n the equilibrium of Sal B–HHb system and Sal B can bind to HHb19]. In other words, an isoactinic point was considered as a directvidence for drug–protein complex formation [23].

.3. The quenching mechanism

Quenching can be classified as either dynamic or static quench-ng by different mechanisms. Dynamic quenching results fromollision between fluorophore and quencher, and static quench-

Part A 78 (2011) 1295–1301 1297

ing is due to the formation of ground-state complex betweenfluorophore and quencher [24]. In general, dynamic and staticquenching can be distinguished by their different dependenceon temperature and viscosity. Dynamic quenching depends upondiffusion. Since higher temperatures result in larger diffusion coef-ficients, the bimolecular quenching constant is expected to increasewith increasing temperature. In contrast, increased temperature islikely to result in decreased stability of complexes and thus leadsto lower values of the static quenching constants.

In order to confirm the quenching mechanism, the fluorescencequenching data are analyzed by the well known Stern–Volmer Eq.(1) [24] and modified Stern–Volmer Eq. (2) [25].

F0

F= 1 + Kq�0[Q ] = 1 + KSV[Q ] (1)

F0

�F= F0

F0 − F= 1

fa+ 1

faKa[Q ](2)

where F0 and F are the fluorescence intensities before and afterthe addition of the quencher, respectively. Kq, KSV, �0, fa, Ka,and [Q] are the quenching rate constant of the biomolecule, theStern–Volmer dynamic quenching constant, the average lifetimeof the biomolecule without quencher (�0 = 10−8 s [26]), the frac-tion of accessible fluorescence, the effective quenching constant forthe accessible fluorophores, and the concentration of the quencher,respectively.

Within certain concentration, the curve of F0/F versus [Q](Stern–Volmer curve) would be linear if the quenching type is singlestatic or dynamic quenching [27]; similarly, the curve of F0/(F0 − F)versus 1/[Q] (modified Stern–Volmer curve) would linear for staticquenching [28]. If the quenching type is combined quenching (bothstatic and dynamic), the Stern–Volmer plot is an upward curvature[29].

Fig. 4(A) displays the Stern–Volmer plots of the quenching ofHHb fluorescence by Sal B. The plot shows that within the investi-gated concentrations, the results agree with the Stern–Volmer Eq.(1). Quenching type should be single static or dynamic quenching.Fig. 4(B) shows the modified Stern–Volmer curves. From Fig. 4(B),it was known that under certain Sal B concentration, the curves ofF0/(F0 − F) versus 1/[Q] were linear. All these results showed thatthere were obviously characters of static quenching.

As a rule, the KSV values decrease with an increase in tem-perature for static quenching, and the reverse effect would beobserved for dynamic quenching [30]; the maximum scatter col-lision quenching constant, Kq,max of various quenchers with thebiopolymer was 2.0 × 1010 L mol−1 s−1 [28]. If the Kq > Kq,max, thefluorescence quenching of biopolymer surely do not come fromdynamic quenching. In this paper, the KSV, and Kq at differenttemperatures were listed in Table 1. It indicated that the KSV val-ues decreased with an increase in temperature and the Kq wasapproximately 1012 L mol−1 s−1. Obviously, this indicated that thequenching was not initiated from dynamic collision but from theformation of a compound. In addition, the corresponding resultsof Ka values were also listed in Table 1. The decreasing trend of Ka

with increasing temperature was in accordance with KSV’s depen-dence on temperature as mentioned above, which also coincideswith static quenching mechanism.

3.4. The binding constants and the number of binding sites

For the static quenching process, when small molecules bindindependently to a set of equivalent sites on a biomacromolecule,

the equilibrium between free and bound molecules is given by thefollowing equation [31]:

logF0 − F

F= log Kb + n log[Q ] (3)

1298 T. Chen et al. / Spectrochimica Acta Part A 78 (2011) 1295–1301

2.52.01.51.00.50.0

0.9

1.2

1.5

1.8

2.1

2.4

2.7

3.0

F0

/F

[Q] (10-5 M)

291 K

300 K

4.54.03.53.02.52.01.51.00.50.0

1

2

3

4

5

6

7

8

9

F0 /

(F

0-F

)

1 / [Q] (105 M

-1)

291 K

300 K

A

B

FqC

wpni(po(1stahctt

-4.6-4.8-5.0-5.2-5.4-5.6

-1.0

-0.8

-0.6

-0.4

-0.2

0.0

0.2

0.4

291 K

300 K

log

10

[(F

0-F

)/F

]

forces and hydrogen-bond formation in low dielectric media. More-

TS

ig. 4. (A) Stern–Volmer curves and (B) modified Stern–Volmer curves for theuenching of HHb fluorescence by Sal B at different temperatures (291 K and 310 K).HHb = 5.0 �M; pH 7.4.

here F0 and F are the fluorescence intensities in the absence andresence of the quencher, respectively, Kb is the binding constant,is the number of binding sites per albumin molecule and [Q]

s the concentration of quencher. Fig. 5 showed the plots of logF0 − F)/F versus log[Q] for the Sal B–HHb system at different tem-eratures, the calculated binding constants (Kb) and the numberf binding sites (n) are presented in Table 2. The binding constantsKb) between Sal B and HHb were 7.05 × 104 (291 K, R = 0.9970) and.31 × 105 L mol−1 (300 K, R = 0.9980), and the number of bindingites (n) was determined as 1.043 ± 0.031. These results illustratedhat there is a strong binding force between Sal B and HHb, andlmost one molecular of Sal B binds to one molecular of HHb with

igh affinity. The correlation coefficients are larger than 0.997, indi-ating that the interaction between Sal B and HHb agrees well withhe site-binding model underlying Eq. (3). The value of Kb illus-rated that there is a strong binding force between Sal B and HHb,

able 1tern–Volmer quenching constant (KSV) and modified Stern–Volmer association constant

T (K) Eq. (1)

10−4 KSV (L mol−1) 10−12 Kq (L mol−1 s−1) Ra

291 6.58 6.58 0.9966300 6.29 6.29 0.9964

a R is the correlation coefficient.b SD is the standard deviation of the fit.

log10 [Q]

Fig. 5. Plots of log10 (F0 − F)/F against log10 [Q] for Sal B quenching effect on HHbfluorescence at different temperatures.

which implies that Sal B can be tightly stored and carried by HHbin the body.

3.5. Thermodynamic parameters and nature of the binding forces

Generally, there are essentially four types of non-covalent inter-action that could play a key role in ligand binding to proteins.These are hydrogen bonds, van der Waals forces, hydrophobicand electrostatic interactions. Thermodynamic parameters, freeenergy (�G), standard enthalpy (�H) and standard entropy (�S)can provide an insight into the binding mode [32]. Among theseparameters, �G reflects the possibly of reaction, �H and �S arethe main evidence to determine acting forces. When temperaturevaries in a small range, the �H can be considered as a constant. Thenthrough the binding constant Kb, thermodynamic parameters areevaluated using the following equations:

�G = −RT ln Kb (4)

lnKb2

Kb1=

[1T1

− 1T2

]�H

R(5)

�G = �H − T�S (6)

where Kb2 and Kb1 are the binding constants at different tempera-tures and R is gas constant. The �H, �G and �S for the interaction ofSal B with HHb at different temperatures are shown in Table 2. Thenegative sign for �G means that the interaction process is sponta-neous. Ross and Subramanian [32] have characterized the sign andmagnitude of the thermodynamic parameter associated with var-ious individual kinds of interaction that may take place in proteinassociation processes. For typical hydrophobic interactions, both�H and �S are positive, while these are negative for van der Waals

over, the specific electrostatic interaction between ionic species inan aqueous solution is characterized by positive �S value and nega-tive �H value (very small, almost zero). In the present case, �H and�S for the binding reaction between Sal B and HHb are found to be

(Ka) of the system of Sal B–HHb at different temperatures.

Eq. (2)

SDb 10−4 Ka (L mol−1) Ra SDb

0.0473 6.56 0.9976 0.12490.0470 5.31 0.9994 0.0798

T. Chen et al. / Spectrochimica Acta Part A 78 (2011) 1295–1301 1299

Table 2Binding constant Kb and relative thermodynamic parameters of the system of Sal B–HHb.

T (K) Kb (L mol−1) n Ra SDb �H (kJ mol−1) �S (J mol−1 K−1) �G (kJ mol−1)

4 02650232

4uSohH

3

altolatlSH

E

wta

R

dattst

FsU

291 7.05 × 10 1.012 0.9970 0.300 1.31 × 105 1.074 0.9980 0.

a R is the correlation coefficient.b SD is the standard deviation of the fit.

9.76 kJ mol−1 and 263.86 J mol−1 K−1. The positive �H and �S val-es indicated that hydrophobic interactions play a major role in theal B–HHb binding reaction and make contributions to the stabilityf the complex. From the structure of Sal B (Fig. 1), the aromaticydrocarbon can easily integrate into the hydrophobic pocket ofHb.

.6. Energy transfer from HHb to Sal B

Fluorescence resonance energy transfer (FRET) has been used as“spectroscopic ruler” for measuring molecular distances in bio-

ogical and macromolecular systems [33]. According to the Förster,he efficiency of FRET between donor and acceptor depends mainlyn the following factors: (i) the donor can produce fluorescenceight; (ii) fluorescence emission spectrum of the donor and UV–visbsorbance spectrum of the acceptor have more overlap; and (iii)he distance between the donor and the acceptor approach and isower than 8 nm [34]. Here the donor and acceptor were HHb andal B, respectively. Using FRET, the distance r between Sal B andHb could be calculated by the following equation [24].

= 1 − F

F0= R6

0

(R60 + r6)

(7)

here E denotes the efficiency of transfer between the donor andhe acceptor, r is the average distances between donor and acceptor,nd R0 is the critical distance when the efficiency of transfer is 50%.

60 = 8.79 × 10−25K2N−4˚J (8)

In Eq. (7), K2 is the orientation related to the geometry of theonor and acceptor of dipoles and K2 = 2/3 for random orientations in fluid solution; N is the average refracted index of medium in

he wavelength range where spectral overlap is significant; Ф ishe fluorescence quantum yield of the donor; J is the effect of thepectral overlap between the emission spectrum of the donor andhe absorption spectrum of the acceptor (Fig. 6), which could be

4204003803603403203000

100

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300

400

500

0.00

0.02

0.04

0.06

0.08

0.10

0.12

0.14

0.16

Flu

ore

sc

en

ce

In

ten

sit

ity

Wavelength (nm)

Ab

so

rban

ce

ig. 6. The overlap of the UV–vis absorption of Sal B with the fluorescence emissionpectrum of HHb: (a) the fluorescence spectrum of HHb, CHHb = 5.0 �M and (b) theV–vis absorbance spectrum of Sal B, CSal B = 15.0 �M (T = 291 K, pH 7.4).

49.76 263.86 −27.02−29.40

calculated by the equation:

J =∫ ∞

0F(�)ε(�)�4d�∫ ∞0

F(�)d�(9)

where F(�) is the corrected fluorescence intensity of the donorin the wavelength range, from � to � + ��; ε(�) is the extinctioncoefficient of the acceptor at �.

In the present case, N = 1.36, ˚ = 0.062 for Hb [35], accord-ing to Eqs. (7)–(9), we could calculate J = 8.31 × 10−15 cm3 L mol−1;E = 0.227; R0 = 2.11 nm; and the binding distance r = 2.64 nm. Theaverage distances between a donor fluorophore and acceptor flu-orophore are on the 2–8 nm scale [36], and 0.5R0 < r < 1.5R0 [37],which indicate that the energy transfer from HHb to Sal B occurswith high probability. In accordance with prediction by Förster’snon-radiative energy transfer theory, these results indicated againa static quenching mechanism between Sal B and HHb.

3.7. Conformation investigation

To further verify the binding of Sal B to HHb and investigatethe effects of the binding on HHb conformation, the methods ofsynchronous fluorescence and circular dichroism were utilized.

3.7.1. Synchronous fluorescence studiesSynchronous fluorescence spectroscopy can provide the infor-

mation about changes in the molecular microenvironment in avicinity of the fluorophore functional groups. According to thetheory of Miller [38], when the scanning interval �� between exci-tation and emission wavelength was stabilized at 15 and 60 nm,respectively, the synchronous fluorescence gives the characteris-tic information of Tyr or Trp residues. When �� is set at 15 or60 nm, the shift of the maximum emission wavelength reveals thealteration of polarity microenvironment around Tyr or Trp residues[39]. By investigating the synchronous fluorescence spectra ofTyr residues and Trp residues, we could explore the microenvi-ronmental changes around the fluorophore groups of HHb. Theeffect of Sal B on the synchronous fluorescence spectra of HHb isshown in Fig. 7. It was shown that the fluorescence intensity ofHHb decreased regularly along with the addition of Sal B, whichfurther demonstrated the occurrence of fluorescence quenchingin the binding process. Moreover, there is no significant shift ofthe maximum emission wavelength with �� = 15 nm (Fig. 7(A))or60 nm (Fig. 7(B)), which implies that interaction of Sal B withHHb does not affect the microenvironment around the Tyr or Trpresidues.

It has been also shown in Fig. 8 that addition of the Sal Bresults in strong fluorescence quenching of Tyr residues of HHb,which decreases about 60.83% in fluorescence intensity, while flu-orescence strength of Trp residues decreases about 60.43%. Thus,there are similar decreasing percentages of fluorescence intensityfor Tyr and Trp residues, indicating that the opportunity of Sal Bapproaching the Try and Trp residues is equal. HHb contains three

Trp residues in each �� dimer, for a total six in the tetramer: two �-14 Trp, two �-15 Trp, and two �-37 Trp. The �-14 Trp and �-15 Trpresidues are outside the submit interface [40], while the �-37 Trpresidue is located at the �1�2 interface, which has been assignedas the primary source of fluorescence emission [20]. The aromatic

1300 T. Chen et al. / Spectrochimica Acta Part A 78 (2011) 1295–1301

340330320310300290280

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Flu

ore

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k

400380360340320300

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400

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Flu

ore

scen

ce In

ten

sit

y

Wavelength (nm)

a

k

Fig. 7. Synchronous fluorescence spectra of interaction between HHb and Sal B (A)ack

rtflo

F�

250240230220210200190

-40

0

40

80

120 HHb

Sal B:HHb=1

Sal B:HHb=3

Sal B:HHb=6

Δεε

/ M

-1c

m-1

Wavelength (nm)

t �� = 15 nm and (B) at �� = 60 nm. Concentration of HHb was 5.0 �M while con-entrations of Sal B were 0, 2.5, 5, 7.5, 10, 12.5, 15, 17.5, 20.0, 22.5, 25 �M from a to(T = 291 K, pH 7.4).

esidues of �-42 Tyr, �-140 Tyr, and �-145 Tyr are also located athe � � interface [41]. The similar decreasing percentages of the

1 2uorescence intensity for Tyr and Trp implies that the binding sitef Sal B on HHb is located at �1�2 interface of HHb.

2.52.01.51.00.50.0

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0 Δλλ = 15 nm

Δλλ = 60 nm

[Q] (10-5

M)

F/F

0

ig. 8. The quenching of HHb synchronous fluorescence by Sal B. C(HHb) = 5 �M. (�)� = 15 nm and (�) �� = 60 nm.

Fig. 9. Far UV-CD spectra of HHb in the absence and presence of increasing con-centration of Sal B in the range of 190–250 nm. CHHb = 5.0 �M, CSal B/CHHb = 0, 1, 3, 6.(T = 291 K, pH 7.4).

3.7.2. CD spectra studiesTo ascertain the possible influence of Sal B binding on the sec-

ondary structure of HHb, CD measurement was also performed inthe absence and presence of Sal B. Consistent with the literature, theCD spectrum for HHb (Fig. 9) monitored in the range 250–190 nmshows the presence of two negative bands at 209 and 222 nm,which are ascribed to the �-helix structure of protein [42]. Thenegative peaks at 209 and 222 nm are both contributed to n → �*transfer for the peptide bond of �-helix [43]. However, Fig. 9 alsoreveals that, for the Sal B–HHb system, the appearance of the CDspectra is exactly similar to that of HHb alone even though the ratioof Sal B to HHb is up to 6:1, which indicates that the structure ofHHb after addition of Sal B was also predominantly �-helix. Thesuperimposed CD spectra of the HHb in the absence and presenceof the Sal B reveal that, at least in the experimental concentrationsrange, there is no perceptible secondary structural change of theHHb upon binding with the Sal B, thus Sal B hardly affects the sec-ondary structure of HHb and can maintain protein stabilization. Thesimilar observations were also reported by Jang et al. [17] and Yuanet al. [44].

4. Conclusions

In summary, the experimental results indicate that Sal B canbind to HHb to form a stable complex with one binding site and thebinding process is spontaneous in which hydrophobic interactionsplay a major role. The synchronous fluorescence results indicatethat interaction of Sal B with HHb does not affect the microenvi-ronment around the Tyr or Trp residues of HHb, and the binding siteof Sal B on HHb may be located at �1�2 interface of HHb. Further-more, CD spectral result demonstrates that Sal B hardly affects thesecondary structure of HHb and can maintain protein stabilization.The binding study of drugs to physiologically important protein Hbis greatly important in pharmacy, pharmacology and biochemistry,which may provide some references for the rational use of drugsin the clinic, and is helpful for clarifying the function of Hb as drugstorage.

Acknowledgements

We are grateful for the financial support from the National Nat-ural Science Foundation of China (Grant No. 20906052), the NaturalScience Foundation of Jiangsu Province (Grant No. BK2010281),and the Science and Technology Bureau of Nantong (Grant No.K2009041).

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